Drug addiction is a chronic relapsing disease of the brain manifested by a variety of behaviors that are detrimental to both the individual and society. An understanding of the neurobiology of addiction will require knowledge about how the brain functions normally and how opioid drugs alter brain functioning over the course of addiction. Opioid drugs initiate their effects by engaging opioid receptors on the cell surface of brain cells. The molecular cloning of cDNAs from the three major subtypes of opioid receptors, mu, delta and kappa, has opened new avenues to study opioid receptor activation and signal transduction pathways. Although predictions about the general nature of the receptor binding site have been made based on the structures of opioid alkaloids and peptides, the role of particular domains and amino acid residues for proper receptor function has been recently studies using the methodology of in vitro mutagenesis in conjunction with analysis of receptor chimeras. This proposal seeks to identify the critical structural determinants that comprise the surface of the ligand binding crevice for the mu and delta receptor, and to identify the residues that are responsible for the ligand selectivity of these receptor subtypes. Our determination that His223 of the mu receptor is critical for ligand recognition and is a possible site for NEM alkylation will be further investigated. The hypothesis that ubiquitination is involved in ligand-dependent endocytosis will be tested. In addition, amino acids within the receptor that are responsible for activation of G proteins and receptor-mediated endocytosis will be characterized. This research will have a direct impact on the long-term objective to understand at the molecular level how opioid receptors interact with their ligands and activate signal transduction pathways that result in cellular responses. It is anticipated that the molecular and cellular studies of the effects of opioid drugs and peptides proposed in this application will aid in the elucidation of the mechanisms involved in tolerance to, and physical dependence on, opioids.